Immunoassay procedures have for many years provided sensitive diagnostic tools for the detection of a variety of substances, generally referred to as ligands. Such procedures are described in a number of articles and texts, an example of which is Reviews on Immunoassay Technology, Ed. S.B. Pal, Pub. Chapman & Hall, 1988.
One type of immunoassay procedure, commonly referred to as ELISA, utilizes a solid support such as the well in a plastic plate in accomplishing the assay. A receptor for a target ligand is bound to the solid support. A liquid sample containing the ligand, having specificity for the bound receptor, is then applied to the plate. Following washing and incubation procedures, an enzyme conjugate having binding specificity for the ligand is added to the well. After further rinsing, a substrate is added which develops color on contact with the bound enzyme, the amount of color developed being dependent upon the amount of bound conjugate present which, in turn, is indicative of the amount of target ligand bound to the support. Thus, by measuring the amount of color development and correlating such against known standards, the concentration of ligand in the sample can be determined. In general, conventional ELISA procedures take on the order of five hours or more.
Recently, it has been suggested that the ELISA procedure can be accelerated by utilizing immunofiltration (Ijsselmuiden et al., Journal of Immunological Method, 119 (1989) 35-43 and Eur. J. Microbiol. 6, (1987) 281). In the disclosed procedure, a nitrocellulose filter is pre-coated with an antigen. Thereafter, a solution containing the target ligand, in this case an antibody, is drawn through the filter followed by rinsing solutions. Finally, either an enzyme-labeled antibody (Ijsselmuiden (1987)) or .sup.125 I-labeled protein A (Ijsselmuiden (1989), both of which have binding specificity for the target ligand, is applied and drawn through the filter to detect the target antibody bound to the antigen on the nitrocellulose filter.
The device used to accomplish the above described assay is illustrated in the 1989 Ijsselmuiden article. It consists of three blocks of perspex which are clamped together during the assay. The bottom section has an external outlet and a valve, and constitutes a reservoir attached to the upper sections. The middle and top sections, designed to accommodate the nitrocellulose filter between them, contain 32 corresponding holes with a diameter of 5 mm and neoprene "O" rings facing the nitrocellulose sheet to prevent lateral flow.
While Ijsselmuiden appears to describe an immunoassay procedure which has the advantage of rapidity over prior procedures, only the procedure utilizing .sup.125 I detection is quantitative. The enzyme-labeled antibody system (Ijsselmuiden (1987)) yields only a qualitative determination of target ligand. A quantitative procedure having the advantages of rapidity and not necessitating the use of radioactively labeled detection reagents such as .sup.125 I would be desirable. In addition, a procedure having the foregoing attributes which also can utilize commercially available microtiter plates and associated readers for determination of color development would be advantageous.